During B cell development, cell surface molecules are expressed or are down regulated and the pattern of expression of these molecules can identify different stages of differentiation or activation. These molecules also provide signals to the B cell for further diferentiation or activation. To generate monoclonal antibodies to different subsets of B cells, hybridomas were produced from C57Bl/6 mice hyperimmunized with DBA/2 spleen cells. One monoclonal antibody recognizes an activation-induced antigen on both B and T cells that is 16-18 kD. This molecule is upregulated on small resting B cells with different stimuli. The combination of anti-IgM, IL4 and IL5 induces its highest density on the B cell surface but it is present on only a small percentage of cells. Individually, these stimuli induce a lower density of antigen on the cell surface but it is expressed on over 50% of the cells. This antibody cross reacts with human cells and is upregulated on human thymocytes cultured with PHA and IL2. Attempts to sequence this antigen and elucidate its function are underway. The ultimate goal of a B cell is to produce antibodies. The mechanism of antibody generation is largely understood in that different gene segments are combined to create an antibody with specificity for a particular antigen. Additional antibody diversity is due to flexibility of joining and somatic mutation. While the recombination process is generally understood, the process of """"""""choosing"""""""" which gene segments to recombine is not. To try to understand these processes, we are studying in detail a small Vk family, Vk10, which is utilized in a number of different antigenic responses in several different murine inbred strains. Levels of mRNA expression of 2 of the members of this family have been quantitated and show a 14.4 fold difference between them. The third member of this family has not been detected in any functional or non-functional light chain to date. Sequence analysis reveals no structural reason preventing the expression of this Vk10 gene. RT-PCR experiments have shown the absence of expression of this gene in the spleens of 3 out of 4 mice. The one mouse to contain mRNA of this Vk10 gene showed extremely low levels of expression. These data suggest that the germline contribution to antibody diversity may not be as great as originally thought.
